Human norovirus (HuNoV) is a major causative agent of foodborne gastroenteritis worldwide. It has been estimated that over 90% of outbreaks of acute nonbacterial gastroenteritis are caused by noroviruses. HuNoV is highly contagious, and only a few particles are thought to be sufficient to cause an infection. Outbreaks frequently occur in restaurants, hotels, daycare centers, schools, nursing homes, cruise ships, swimming pools, hospitals, and in military installations. For these reasons, HuNoV is classified as NIAID Category B priority bio-defense pathogens. Human norrovirus cannot grow in cell culture and there is no small animal model for infection study. Currently, there are no vaccines or effective therapeutic interventions for this virus. Therefore, there is critical and urgent need to develop an effective vaccine against human norovirus.
HuNoV is a non-enveloped, positive-sense RNA virus. The genome of HuNoV contains 7.3-7.7 kb encoding three open reading frames (ORF). ORF1 encodes a polyprotein that is cleaved to produce 6 nonstructural proteins, including the RNA-dependent RNA polymerase (RdRp). ORF2 encodes the major capsid protein (VP1) that contains the antigenic and receptor binding sites. ORF3 encodes a minor capsid protein (VP2) that may play a role in stabilizing virus particles. It is known that the expression of VP1 alone in cell culture yields self-assembled virus-like particles (VLPs) that are structurally and antigenically similar to native virions. Consequently, most HuNoV vaccine studies have focused on VLPs. To date, HuNoV VLPs have been expressed in E. coli, yeast, insect cells, mammalian cell lines, tobacco, and potatoes. Immunization with VLPs orally or intranasally induced variable humoral, mucosal, and cellular immunities. Although these studies are very promising, there are several limitations of developing in vitro-expressed VLPs into a vaccine candidate. Preparation of VLPs in vitro is time consuming and expensive. Immunization usually requires high dosage of VLPs (usually more than 100 μg) and multiple booster immunizations. The efficacy of VLP-based vaccines relies on the addition of mucosal adjuvants such as cholera toxin (CT) and E. coli toxin (LT), which are potentially toxic to the central nervous system. Also, the duration of the antigen stimulation may be limited because VLPs are actually proteins, a non-replicating immunogen.
Generally, a live attenuated vaccine stimulates strong systemic immunity and provides durable protection because replication in vivo results in high level intracellular synthesis of the full complement of viral antigens over a prolonged period. However, such a vaccine is not realistic for viruses that cannot grow in cell culture. Without the ability to grow in cell cultures, the virus cannot be attenuated, and even if an attenuated strain is available, it could not be mass produced. In this situation, a vectored vaccine may be ideal to overcome this obstacle.
VSV is a non-segmented negative-sense (NNS) RNA virus that belongs to virus family Rhabdoviridae. To date, VSV has been examined as a vaccine candidate for a number of pathogens including human immunodeficiency virus (HIV), severe acute respiratory syndrome (SARS), hepatitis C virus, influenza virus, human papillomavirus, measles virus, Ebola virus and Marburg virus. These studies have shown that VSV-based vaccines triggered immunity in animal models. It has been reported that VSV-based HIV vaccine proved efficacious in monkeys and is now in clinical trials. However, the exploration of VSV as a vector to deliver vaccines against non-cultivable foodborne viruses has not been reported.
Since the establishment of the reverse genetics system for VSV in 1995, hundreds of exogenous genes have been expressed by VSV as a vector. However, the feasibility of using VSV as the vector to express and deliver VLPs is poorly understood. To date, there has only been one report which demonstrated using VSV to generate VLPs. Specifically, it was demonstrated that the expression of the hepatitis C virus (HCV) core, E1, and E2 proteins by VSV assembled to form HCV-like particles in BHK-21 cells which was similar to the ultrastructural properties of HCV virions. However, Blanchard et al., (2003) argued that these particles may be the endogenous viruses of BHK-21 cells such as intracisternal Rtype particles, but not the complete budded HCV-like particles. Later, it was shown that expression of HCV E1 and E2 by propagating and non-propagating (G protein deleted) VSV vector resulted in correctly folded E1/E2 heterodimers. However, detailed characterization of HCV-like particles was lacking in their study.
To date, most studies have focused on HuNoV VLPs purified from the baculovirus expression system. It has been shown that HuNoV VLP vaccination induced humoral and cellular immune in both humans and mice. Two live viral vectors, Venezuelan Equine Encephalitis (VEE) and adenovirus, have been studied to deliver HuNoV VLPs. It was shown that the VEE replicon expressing HuNoV VLPs induced HuNoV specific systemic, mucosal, and heterotypic immunity in mice. Using cultivable murine norovirus (MNV) as a model, it was shown that VEE-based vaccine induced homotypic and heterotypic humoral and cellular immunity, and protected mice from MNV challenge.
Most recently, it was reported that a recombinant adenovirus expressing HuNoV capsid protein stimulated a specific immune response in mice.
However, there are some potential disadvantages using VEE and adenovirus as vector. Although VEE replicon is a single cycle replicating vectors, the biosafety of VEE has been questioned since VEE is a biodefense pathogen and the use of functional VEE genes is restricted.
For adenovirus, in vivo delivery of the vectored vaccine may be hampered by the host immune response since the host may have been exposed to adenovirus before.